Journal
ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY
Volume 70, Issue -, Pages 2152-2162Publisher
INT UNION CRYSTALLOGRAPHY
DOI: 10.1107/S139900471401267X
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Funding
- BBSRC [BB/E001084, BB/FOF/263]
- Cardiff Partnership Award
- BBSRC CASE studentship
- Merck KGaA
- BBSRC [BB/E007384/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/E007384/1] Funding Source: researchfish
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Single-amino-acid deletions are a common part of the natural evolutionary landscape but are rarely sampled during protein engineering owing to limited and prejudiced molecular understanding of mutations that shorten the protein backbone. Single-amino-acid deletion variants of enhanced green fluorescent protein (EGFP) have been identified by directed evolution with the beneficial effect of imparting increased cellular fluorescence. Biophysical characterization revealed that increased functional protein production and not changes to the fluorescence parameters was the mechanism that was likely to be responsible. The structure EGFP(D190 Delta) containing a deletion within a loop revealed propagated changes only after the deleted residue. The structure of EGFP(A227 Delta) revealed that a 'flipping' mechanism was used to adjust for residue deletion at the end of a beta-strand, with amino acids C-terminal to the deletion site repositioning to take the place of the deleted amino acid. In both variants new networks of short-range and long-range interactions are generated while maintaining the integrity of the hydrophobic core. Both deletion variants also displayed significant local and long-range changes in dynamics, as evident by changes in B factors compared with EGFP. Rather than being detrimental, deletion mutations can introduce beneficial structural effects through altering core protein properties, folding and dynamics, as well as function.
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